siRNA compositions that specifically down regulate expression of a variant of the PNPLA3 gene and methods of use thereof for treating a chronic liver disease

ABSTRACT

The invention provides siRNA compositions that specifically downregulates expression of a variant of the PNPLA3 gene and methods of use thereof for treating a chronic liver disease or alcoholic liver disease (ALD).

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/026,609, filed Jul. 3, 2017, which is a continuation of U.S. patentapplication Ser. No. 15/614,147, filed Jun. 5, 2017, now issued as U.S.Pat. No. 10,036,024, which claims priority to, and the benefit of, U.S.Provisional Patent Application Ser. No. 62/345,048, filed Jun. 3, 2016,the contents of each of which are incorporated by reference.

FIELD OF THE INVENTION

The invention provides siRNA compositions that specifically regulateexpression of a variant of the PNPLA3 gene and methods of use thereoffor treating a chronic liver disease or alcoholic liver disease (ALD).

BACKGROUND

Nonalcoholic Fatty Liver Disease (NAFLD) is a spectrum of chronic liverdisorders, beginning as hepatic fat accumulation without significantalcohol consumption. A subset of patients with NAFLD have nonalcoholicsteatohepatitis (NASH) which, over time and without treatment, mayprogress to cirrhosis and even hepatocellular carcinoma (HCC). It isestimated that 4-22% of HCC cases in the US are due to NASH, and about2% of the U.S. population has NASH-derived cirrhosis, which is expectedto become the leading cause of liver transplantation by 2020. Moreover,NAFLD/NASH is the central hallmark of obesity and type II diabetes whichtogether affect over 50% of the US population, leading to a heavyeconomic burden. Unfortunately, therapeutic options for NASH are stillvery limited thus far, with only slight benefits observed from vitamin Eor obeticholic acid treatment. Developing safe and effective treatmentsfor NASH remain a significant unmet medical need.

SUMMARY

The invention recognizes that the patatin-like phospholipase domaincontaining 3 (PNPLA3) gene is strongly associated with chronic humanliver disease (e.g., fatty liver disease, steatohepatitis, cirrhosis,alcoholic liver disease and hepatocellular carcinoma). Particularly, the148 I>M (rs738409 C>G) mutation has been identified as the causal allelefor these phenotypes. Overexpression of the 148M isoform is demonstratedherein to be a major cause of these pathogenic processes in both humanhepatocytes and animal models. The invention provides small interferingRNA (siRNA) that can specifically recognize the mutant allele (148M)while having minimal effect on the wild-type allele (148I). In thatmanner, a novel therapeutic strategy for treating human chronic liverdisease due to the overexpression of PNPLA3 148M isoform is provided.

In certain aspects, the invention provides compositions that include asmall interfering RNA (siRNA) molecule that specifically binds mRNAcorresponding to the rs738409 C>G variant of the patatin-likephospholipase domain-containing (PNPLA3) gene, thereby downregulatingexpression of mutant allele. The rs738409 C>G variant is also referredthroughout as the 148M mutant allele or isoform, as opposed to the 148wild-type allele or isoform. The siRNA molecule may be single strandedor double stranded. In certain embodiments, the siRNA molecule consistsof the nucleotide sequence of at least one of SEQ ID NOs 1, 2 or278-552. In certain embodiments, the siRNA molecule does not bind mRNAassociated with the wild-type isoform of the PNPLA3 gene. In otherembodiments, the siRNA molecule includes one or more non-naturallyoccurring nucleotides.

To facilitate effective delivery, the siRNA may be coupled to apharmaceutically acceptable carrier system. In certain embodiments thepharmaceutically acceptable carrier system includes a nanoparticle towhich the siRNA molecule is coupled. An exemplary nanoparticle includeslow molecular weight polyethyleneimine (LPEI) or its derivatives (e.g.,disulfide crosslinked polyethyleneimine (CLPEI)) and a lipid. In certainembodiments, the lipid is a bile acid, such as cholic acid, deoxycholicacid, and lithocholic acid.

Other aspects of the invention provide methods for treating a subjectwith a chronic liver disease that involve administrating atherapeutically effective amount of any of the above compositions to asubject having a chronic liver disease. Exemplary chronic liver diseasesinclude fatty liver disease, steatohepatitis, cirrhosis, alcoholic liverdisease (ALD), or hepatocellular carcinoma.

Other aspects of the invention provide an allele-specific DNA-basedantisense oligo to downregulate expression of the 148M allele.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequence of wild type patatin-like phospholipase domaincontaining 3 (PNPLA3) gene as well as the sequence of the rs738409 C>G(I148M) variant of the PNPLA3 gene.

FIG. 2A shows a schematic of lipid-grafted LPEI preparation.

FIG. 2B shows 1H-NMR analysis of a lipid-grafted LPEI preparation.

FIG. 3A shows the specificity and potency of 148MSi in targetingPNPLA3148M as compared to PNPLA3 148M with transient transfection ofsiRNA and PNPLA3-Luc vectors into HEK293 cells for 48 h. FIGS. 3B and 3Cshow downregulation of endogenous transcription of PNPLA3 in HepG2 (148Mhomozygote) and HEK293 (148I homozygote) cells transfected with 20pmole/ml 148MSi, control (Ctr-Si) or generic PNPLA3 siRNA set (Gen-Si,Santa Cruz) for 48 h. FIGS. 3D-E show reduction of intracellulartriglycerides (TG) accumulation (ORO staining) by 148MSi targeting.After transfection, Huh-7 (148M homozygote) cells were co-incubated withfree fatty acids [palmitic acid (0.3 mM) and oleic acid (0.9 mM)] andglucose (Glu) at 1 mM or 10 mM concentration for 48 h. *p<0.05;**p<0.01.

FIGS. 4A-C shows transfection efficiency of LPEI- or LCA-LPEI polyplexesin NIH-3T3 cells. Cells were seeded at a density of 20,000 cells perwell in a 24-well plate. After 2 days, cells in each well were treatedwith polyplexes consisting of pEGFP-C1 plasmid (0.2 μg) and polymers in10/1 to 20/1 weight ratios±dermatan sulfate (DS, 0.2 μg) and incubatedfor 48 h. GFP expression was visualized with fluorescence microscope(top panel) and quantified by measuring the fluorescence of thesupernatant of cell lysate.

FIG. 5 shows that genetic variation in PNPLA3 confers susceptibility tochronic liver disease.

FIG. 6 shows differences between the 148I phenotype and the 148Mphenotype.

FIG. 7 shows vectors for compositions of the invention and theireffectiveness in downregulating the 148M phenotype.

FIGS. 8A-D show in vivo effectiveness of compositions of the inventionin reducing hepatic PNPLA3^(148M) expression and total reduction oftotal hepatic TG levels.

DETAILED DESCRIPTION

The invention provides siRNA compositions that specifically downregulateexpression of a variant of the PNPLA3 gene and methods of use thereoffor treating chronic liver disease. In certain aspects, the inventionprovides compositions including a small interfering RNA (siRNA) moleculethat specifically binds mRNA transcribed from a rs738409 C>G variant ofa patatin-like phospholipase domain-containing (PNPLA3) gene.

The patatin-like phospholipase domain-containing (PNPLA3) gene refers toNCBI Gene ID: 80339. FIG. 1 shows the sequence of wild type patatin-likephospholipase domain-containing (PNPLA3) gene as well as the sequence ofthe rs738409 C>G (I148M) variant of the PNPLA3 gene. Genome-wideassociation studies (GWAS) in recent years have identified the rs738409C>G (I148M) variant of the patatin-like phospholipase domain-containing3 (PNPLA3) gene as the strongest genetic risk allele for NAFLD/NASH,influencing degree of steatosis, grade of inflammation, stage offibrosis and risk of HCC among all examined populations (FIG. 5). Otherstudies have also demonstrated the strong association between 148Mallele and ALD (Buch et al., (Nature Genetics, 47(12):1443-1447, 2015),the content of which is incorporated by reference herein in itsentirety). Notably, NAFLD patients and diabetic patients carrying the148M mutant allele have over 12- and 19-fold higher risk for thedevelopment of HCC, respectively, as compared to those who are 148Icarriers, making this mutant allele the single largest genetic riskfactor for HCC in the context of NASH. See Liu et al. (Journal ofHepatology, 61:75-81, 2014), the content of which is incorporated byreference herein in its entirety. The 148M allele frequency varies from˜12% among African decedents, 24-40% among Caucasian and East Asianpopulations to ˜50% among Hispanic populations, accounting for a largevariability in genetic susceptibility to NAFLD/NASH. Thus far, a numberof mechanistic studies have validated the role of the PNPLA3 148M allelein the development of NAFLD. Accordingly, the present inventionrecognizes that targeting PNPLA3 may provide an ideal therapeutic optionfor the treatment of NAFLD/NASH. To date, no drug has been developed totarget PNPLA3.

While the detailed molecular mechanism underlying the causal role ofPNPLA3148M in NAFLD/NASH/ALD still remains incompletely understood, bothin vitro cell line studies and in vivo studies using animal models haveconsistently demonstrated that induction of NAFLD phenotypes requires an“dominant-negative” effect of PNPLA3 (i.e., overexpression of PNPLA3148M rather than PNPLA3 148I or gene deletion). More specifically, ithas been validated that PNPLA3 148M leads to a loss-of-function of itstriglycerides hydrolysis activity. However, knockout of the PNPLA3 genein mice does not lead to NAFLD phenotypes. Overexpression of PNPLA3 148Mrather than PNPLA3 148I have been found to induce NAFLD. Moreover,inducing the expression of a PNPLA3 catalytic activity-negative mutant,S47A in a knock-in mouse model parallels the effect of PNPAL3 148M,further highlighting the essential role of the high transcription levelof PNPLA3 148M isoform in the development of NAFLD. Given theloss-of-function nature of 148M isoform, conventional therapeuticstrategies (e.g., to develop agonist or antagonist chemicals targetingthe PNPLA3 protein) are unlikely to block the pathogenic effect ofPNPLA3 148M. Instead, specifically reducing the transcription ofPNPLA3148M shows great potential.

The invention recognizes that due to its first-pass extract effect, theliver is the organ with the most successful siRNA delivery. Accordingly,the invention provides RNAi-based therapeutics targeting PNPLA3,especially with a capability of allele-specific downregulation of 148Mtranscription. In certain embodiments, it may be found that the siRNAmolecules of the invention are highly specific and potent indownregulating expression of the PNPLA3 148M allele without effectingexpression of the PNLPLA3 148I wild-type allele. In certain embodiments,novel nanoparticles capable of siRNA delivery may be used.

The siRNA molecules within the compositions of the inventionspecifically bind mRNA transcribed from the PNPLA3 148M allele. Here,the term specific or specifically, used in combination with e.g.,binding, hybridization, or downregulating refers to binding of a targetsequence or downregulation of a target gene's expression with minimal orno binding or downregulation of other nucleic acids or their expression.In particular, mRNA transcribed from the PNPLA3 148I allele (wild-type)is not bound and expression of wild-type PNPLA3 is not downregulated bysiRNA of the invention that specifically downregulates expression of thePNPLA3 148M allele. Specific binding as used herein may refer to siRNAthat hybridize to a target mRNA sequence under high stringencyconditions. Nucleic acid hybridization may be affected by suchconditions as salt concentration, temperature, or organic solvents, inaddition to base composition, length of complementary strands, andnumber of nucleotide base mismatches between hybridizing nucleic acids,as is readily appreciated by those skilled in the art. Stringency ofhybridization reactions is readily determinable by one of ordinary skillin the art, and generally is an empirical calculation dependent uponsequence length, washing temperature, and salt concentration. Ingeneral, longer sequences require higher temperatures for properannealing, while shorter sequences need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowits melting temperature. The higher the degree of desired homologybetween the sequence and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995), the contents of which are incorporated by referenceherein in their entirety.

Stringent conditions or high stringency conditions typically: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1%SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C.

Moderately stringent conditions may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989 (the contents of which are incorporated byreference herein in their entirety), and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent that those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37° C. to 50° C. Theskilled artisan will recognize how to adjust the temperature, ionicstrength, etc. as necessary to accommodate factors such as sequencelength and the like.

Interfering RNA (which may be interchangeably referred to as RNAi or aninterfering RNA sequence) refers to double-stranded RNA that is capableof silencing, reducing, or inhibiting expression of a target gene by anymechanism of action now known or yet to be disclosed. For example, RNAimay act by mediating the degradation of mRNAs which are complementary tothe sequence of the RNAi when the RNAi is in the same cell as the targetgene. As used herein, RNAi may refer to double-stranded RNA formed bytwo complementary RNA strands or by a single, self-complementary strand.RNAi may be substantially or completely complementary to the target mRNAor may comprise one or more mismatches upon alignment to the targetmRNA. The sequence of the interfering RNA may correspond to the fulllength target mRNA, or any subsequence thereof.

The concept of RNAi includes small-interfering RNA, which, herein, mayinterchangeably be referred to as siRNA. siRNA is described for examplein U.S. Pat. Nos. 9,328,347; 9,328,348; 9,289,514; 9,289,505; and9,273,312, the content of each of which is incorporated by referenceherein in its entirety. A siRNA may be any interfering RNA with a duplexlength of about 15-60, 15-50, or 15-40 nucleotides in length, moretypically about 15-30, 15-25, or 18-23 nucleotides in length. Eachcomplementary sequence of the double-stranded siRNA may be 15-60, 15-50,15-40, 15-30, 15-25, or 18-23 nucleotides in length, but othernoncomplementary sequences may be present. For example, siRNA duplexesmay comprise 3′ overhangs of 1 to 4 or more nucleotides and/or 5′phosphate termini comprising 1 to 4 or more nucleotides. A siRNA may besynthesized in any of a number of conformations. One skilled in the artwould recognize the type of siRNA conformation to be used for aparticular purpose. Examples of siRNA conformations include, but neednot be limited to, a double-stranded polynucleotide molecule assembledfrom two separate stranded molecules, wherein one strand is the sensestrand and the other is the complementary antisense strand; adouble-stranded polynucleotide molecule assembled from a single-strandedmolecule, where the sense and antisense regions are linked by a nucleicacid-based or non-nucleic acid-based linker; a double-strandedpolynucleotide molecule with a hairpin secondary structure havingcomplementary sense and antisense regions; or a circular single-strandedpolynucleotide molecule with two or more loop structures and a stemhaving self-complementary sense and antisense regions. In the case ofthe circular polynucleotide, the polynucleotide may be processed eitherin vivo or in vitro to generate an active double-stranded siRNAmolecule.

SiRNA can be chemically synthesized, may be encoded by a plasmid andtranscribed, or may be vectored by a virus engineered to express thesiRNA. A siRNA may be a single stranded molecule with complementarysequences that self-hybridize into duplexes with hairpin loops. siRNAcan also be generated by cleavage of parent dsRNA through the use of anappropriate enzyme such as E. coli RNase III or Dicer (Yang et al, Proc.Natl. Acad. Sci. USA 99, 9942-9947 (2002); Calegari et al, Proc. Natl.Acad. Sci. USA 99, 14236-14240 (2002); Byrom et al, Ambion TechNotes 10,4-6 (2003); Kawasaki et al, Nucleic Acids Res 31, 981-987 (2003); Knightet al, Science 293, 2269-2271 (2001); and Robertson et al, J Biol Chem243, 82-91 (1968)). A parent dsRNA may be any double stranded RNA duplexfrom which a siRNA may be produced, such as a full or partial mRNAtranscript.

A mismatch motif may be any portion of a siRNA sequence that is not 100%complementary to its target sequence. A siRNA may have zero, one, two,or three or more mismatch regions. The mismatch regions may becontiguous or may be separated by any number of complementarynucleotides. The mismatch motifs or regions may comprise a singlenucleotide or may comprise two or more consecutive nucleotides.

A preferred example of a double stranded siRNA design of the inventionis listed in Table 1:

TABLE 1 1 5′-CCUGCUUCAUGCCUUUCUACAGUGG-3′ 23′-AAGGACGAAGUACGGAAAGAUGUCACC-5′

Exemplary DNA targets for siRNA molecules or antisense oligonucleotidesof the invention may include the sequences listed in Table 2 below.

TABLE 2 SEQ ID NO DNA target Sequence   3 GGCCTTGGTATGTTCCTGCTTCATG   4GCCTTGGTATGTTCCTGCTTCATG   5 CCTTGGTATGTTCCTGCTTCATG   6CTTGGTATGTTCCTGCTTCATG   7 TTGGTATGTTCCTGCTTCATG   8TGGTATGTTCCTGCTTCATG   9 GGTATGTTCCTGCTTCATG  10 GTATGTTCCTGCTTCATG  11TATGTTCCTGCTTCATG  12 ATGTTCCTGCTTCATG  13 TGTTCCTGCTTCATG  14GCCTTGGTATGTTCCTGCTTCATGC  15 CCTTGGTATGTTCCTGCTTCATGC  16CTTGGTATGTTCCTGCTTCATGC  17 TTGGTATGTTCCTGCTTCATGC  18TGGTATGTTCCTGCTTCATGC  19 GGTATGTTCCTGCTTCATGC  20 GTATGTTCCTGCTTCATGC 21 TATGTTCCTGCTTCATGC  22 ATGTTCCTGCTTCATGC  23 TGTTCCTGCTTCATGC  24GTTCCTGCTTCATGC  25 CCTTGGTATGTTCCTGCTTCATGCC  26CTTGGTATGTTCCTGCTTCATGCC  27 TTGGTATGTTCCTGCTTCATGCC  28TGGTATGTTCCTGCTTCATGCC  29 GGTATGTTCCTGCTTCATGCC  30GTATGTTCCTGCTTCATGCC  31 TATGTTCCTGCTTCATGCC  32 ATGTTCCTGCTTCATGCC  33TGTTCCTGCTTCATGCC  34  GTTCCTGCTTCATGCC  35 TTCCTGCTTCATGCC  36CTTGGTATGTTCCTGCTTCATGCCT  37 TTGGTATGTTCCTGCTTCATGCCT  38TGGTATGTTCCTGCTTCATGCCT  39 GGTATGTTCCTGCTTCATGCCT  40GTATGTTCCTGCTTCATGCCT  41 TATGTTCCTGCTTCATGCCT  42 ATGTTCCTGCTTCATGCCT 43 TGTTCCTGCTTCATGCCT  44 GTTCCTGCTTCATGCCT  45 TTCCTGCTTCATGCCT  46TCCTGCTTCATGCCT  47 TTGGTATGTTCCTGCTTCATGCCTT  48TGGTATGTTCCTGCTTCATGCCTT  49 GGTATGTTCCTGCTTCATGCCTT  50GTATGTTCCTGCTTCATGCCTT  51 TATGTTCCTGCTTCATGCCTT  52ATGTTCCTGCTTCATGCCTT  53 TGTTCCTGCTTCATGCCTT  54 GTTCCTGCTTCATGCCTT  55TTCCTGCTTCATGCCTT  56 TCCTGCTTCATGCCTT  57 CCTGCTTCATGCCTT  58TGGTATGTTCCTGCTTCATGCCTTT  59 GGTATGTTCCTGCTTCATGCCTTT  60GTATGTTCCTGCTTCATGCCTTT  61 TATGTTCCTGCTTCATGCCTTT  62ATGTTCCTGCTTCATGCCTTT  63 TGTTCCTGCTTCATGCCTTT  64 GTTCCTGCTTCATGCCTTT 65 TTCCTGCTTCATGCCTTT  66 TCCTGCTTCATGCCTTT  67 CCTGCTTCATGCCTTT  68CTGCTTCATGCCTTT  69 GGTATGTTCCTGCTTCATGCCTTTC  70GTATGTTCCTGCTTCATGCCTTTC  71 TATGTTCCTGCTTCATGCCTTTC  72ATGTTCCTGCTTCATGCCTTTC  73 TGTTCCTGCTTCATGCCTTTC  74GTTCCTGCTTCATGCCTTTC  75 TTCCTGCTTCATGCCTTTC  76 TCCTGCTTCATGCCTTTC  77CCTGCTTCATGCCTTTC  78 CTGCTTCATGCCTTTC  79 TGCTTCATGCCTTTC  80GTATGTTCCTGCTTCATGCCTTTCT  81 TATGTTCCTGCTTCATGCCTTTCT  82ATGTTCCTGCTTCATGCCTTTCT  83 TGTTCCTGCTTCATGCCTTTCT  84GTTCCTGCTTCATGCCTTTCT  85 TTCCTGCTTCATGCCTTTCT  86 TCCTGCTTCATGCCTTTCT 87 CCTGCTTCATGCCTTTCT  88 CTGCTTCATGCCTTTCT  89 TGCTTCATGCCTTTCT  90GCTTCATGCCTTTCT  91 TATGTTCCTGCTTCATGCCTTTCTA  92ATGTTCCTGCTTCATGCCTTTCTA  93 TGTTCCTGCTTCATGCCTTTCTA  94GTTCCTGCTTCATGCCTTTCTA  95 TTCCTGCTTCATGCCTTTCTA  96TCCTGCTTCATGCCTTTCTA  97 CCTGCTTCATGCCTTTCTA  98 CTGCTTCATGCCTTTCTA  99TGCTTCATGCCTTTCTA 100 GCTTCATGCCTTTCTA 101 CTTCATGCCTTTCTA 102ATGTTCCTGCTTCATGCCTTTCTAC 103 TGTTCCTGCTTCATGCCTTTCTAC 104GTTCCTGCTTCATGCCTTTCTAC 105 TTCCTGCTTCATGCCTTTCTAC 106TCCTGCTTCATGCCTTTCTAC 107 CCTGCTTCATGCCTTTCTAC 108 CTGCTTCATGCCTTTCTAC109 TGCTTCATGCCTTTCTAC 110 GCTTCATGCCTTTCTAC 111 CTTCATGCCTTTCTAC 112TTCATGCCTTTCTAC 113 TGTTCCTGCTTCATGCCTTTCTACA 114GTTCCTGCTTCATGCCTTTCTACA 115 TTCCTGCTTCATGCCTTTCTACA 116TCCTGCTTCATGCCTTTCTACA 117 CCTGCTTCATGCCTTTCTACA 118CTGCTTCATGCCTTTCTACA 119 TGCTTCATGCCTTTCTACA 120 GCTTCATGCCTTTCTACA 121CTTCATGCCTTTCTACA 122 TTCATGCCTTTCTACA 123 TCATGCCTTTCTACA 124GTTCCTGCTTCATGCCTTTCTACAG 125 TTCCTGCTTCATGCCTTTCTACAG 126TCCTGCTTCATGCCTTTCTACAG 127 CCTGCTTCATGCCTTTCTACAG 128CTGCTTCATGCCTTTCTACAG 129 TGCTTCATGCCTTTCTACAG 130 GCTTCATGCCTTTCTACAG131 CTTCATGCCTTTCTACAG 132 TTCATGCCTTTCTACAG 133 TCATGCCTTTCTACAG 134CATGCCTTTCTACAG 135 TTCCTGCTTCATGCCTTTCTACAGT 136TCCTGCTTCATGCCTTTCTACAGT 137 CCTGCTTCATGCCTTTCTACAGT 138CTGCTTCATGCCTTTCTACAGT 139 TGCTTCATGCCTTTCTACAGT 140GCTTCATGCCTTTCTACAGT 141 CTTCATGCCTTTCTACAGT 142 TTCATGCCTTTCTACAGT 143TCATGCCTTTCTACAGT 144 CATGCCTTTCTACAGT 145 ATGCCTTTCTACAGT 146TCCTGCTTCATGCCTTTCTACAGTG 147 CCTGCTTCATGCCTTTCTACAGTG 148CTGCTTCATGCCTTTCTACAGTG 149 TGCTTCATGCCTTTCTACAGTG 150GCTTCATGCCTTTCTACAGTG 151 CTTCATGCCTTTCTACAGTG 152 TTCATGCCTTTCTACAGTG153 TCATGCCTTTCTACAGTG 154 CATGCCTTTCTACAGTG 155 ATGCCTTTCTACAGTG 156TGCCTTTCTACAGTG 157 CCTGCTTCATGCCTTTCTACAGTGG 158CTGCTTCATGCCTTTCTACAGTGG 159 TGCTTCATGCCTTTCTACAGTGG 160GCTTCATGCCTTTCTACAGTGG 161 CTTCATGCCTTTCTACAGTGG 162TTCATGCCTTTCTACAGTGG 163 TCATGCCTTTCTACAGTGG 164 CATGCCTTTCTACAGTGG 165ATGCCTTTCTACAGTGG 166 TGCCTTTCTACAGTGG 167 GCCTTTCTACAGTGG 168CTGCTTCATGCCTTTCTACAGTGGC 169 TGCTTCATGCCTTTCTACAGTGGC 170GCTTCATGCCTTTCTACAGTGGC 171 CTTCATGCCTTTCTACAGTGGC 172TTCATGCCTTTCTACAGTGGC 173 TCATGCCTTTCTACAGTGGC 174 CATGCCTTTCTACAGTGGC175 ATGCCTTTCTACAGTGGC 176 TGCCTTTCTACAGTGGC 177 GCCTTTCTACAGTGGC 178CCTTTCTACAGTGGC 179 TGCTTCATGCCTTTCTACAGTGGCC 180GCTTCATGCCTTTCTACAGTGGCC 181 CTTCATGCCTTTCTACAGTGGCC 182TTCATGCCTTTCTACAGTGGCC 183 TCATGCCTTTCTACAGTGGCC 184CATGCCTTTCTACAGTGGCC 185 ATGCCTTTCTACAGTGGCC 186 TGCCTTTCTACAGTGGCC 187GCCTTTCTACAGTGGCC 188 CCTTTCTACAGTGGCC 189 CTTTCTACAGTGGCC 190GCTTCATGCCTTTCTACAGTGGCCT 191 CTTCATGCCTTTCTACAGTGGCCT 192TTCATGCCTTTCTACAGTGGCCT 193 TCATGCCTTTCTACAGTGGCCT 194CATGCCTTTCTACAGTGGCCT 195 ATGCCTTTCTACAGTGGCCT 196 TGCCTTTCTACAGTGGCCT197 GCCTTTCTACAGTGGCCT 198 CCTTTCTACAGTGGCCT 199 CTTTCTACAGTGGCCT 200TTTCTACAGTGGCCT 201 CTTCATGCCTTTCTACAGTGGCCTT 202TTCATGCCTTTCTACAGTGGCCTT 203 TCATGCCTTTCTACAGTGGCCTT 204CATGCCTTTCTACAGTGGCCTT 205 ATGCCTTTCTACAGTGGCCTT 206TGCCTTTCTACAGTGGCCTT 207 GCCTTTCTACAGTGGCCTT 208 CCTTTCTACAGTGGCCTT 209CTTTCTACAGTGGCCTT 210 TTTCTACAGTGGCCTT 211 TTCTACAGTGGCCTT 212TTCATGCCTTTCTACAGTGGCCTTA 213 TCATGCCTTTCTACAGTGGCCTTA 214CATGCCTTTCTACAGTGGCCTTA 215 ATGCCTTTCTACAGTGGCCTTA 216TGCCTTTCTACAGTGGCCTTA 217 GCCTTTCTACAGTGGCCTTA 218 CCTTTCTACAGTGGCCTTA219 CTTTCTACAGTGGCCTTA 220 TTTCTACAGTGGCCTTA 221 TTCTACAGTGGCCTTA 222TCTACAGTGGCCTTA 223 TCATGCCTTTCTACAGTGGCCTTAT 224CATGCCTTTCTACAGTGGCCTTAT 225 ATGCCTTTCTACAGTGGCCTTAT 226TGCCTTTCTACAGTGGCCTTAT 227 GCCTTTCTACAGTGGCCTTAT 228CCTTTCTACAGTGGCCTTAT 229 CTTTCTACAGTGGCCTTAT 230 TTTCTACAGTGGCCTTAT 231TTCTACAGTGGCCTTAT 232 TCTACAGTGGCCTTAT 233 CTACAGTGGCCTTAT 234CATGCCTTTCTACAGTGGCCTTATC 235 ATGCCTTTCTACAGTGGCCTTATC 236TGCCTTTCTACAGTGGCCTTATC 237 GCCTTTCTACAGTGGCCTTATC 238CCTTTCTACAGTGGCCTTATC 239 CTTTCTACAGTGGCCTTATC 240 TTTCTACAGTGGCCTTATC241 TTCTACAGTGGCCTTATC 242 TCTACAGTGGCCTTATC 243 CTACAGTGGCCTTATC 244TACAGTGGCCTTATC 245 ATGCCTTTCTACAGTGGCCTTATCC 246TGCCTTTCTACAGTGGCCTTATCC 247 GCCTTTCTACAGTGGCCTTATCC 248CCTTTCTACAGTGGCCTTATCC 249 CTTTCTACAGTGGCCTTATCC 250TTTCTACAGTGGCCTTATCC 251 TTCTACAGTGGCCTTATCC 252 TCTACAGTGGCCTTATCC 253CTACAGTGGCCTTATCC 254 TACAGTGGCCTTATCC 255 ACAGTGGCCTTATCC 256TGCCTTTCTACAGTGGCCTTATCCC 257 GCCTTTCTACAGTGGCCTTATCCC 258CCTTTCTACAGTGGCCTTATCCC 259 CTTTCTACAGTGGCCTTATCCC 260TTTCTACAGTGGCCTTATCCC 261 TTCTACAGTGGCCTTATCCC 262 TCTACAGTGGCCTTATCCC263 CTACAGTGGCCTTATCCC 264 TACAGTGGCCTTATCCC 265 TCAGTGGCCTTATCCC 266CAGTGGCCTTATCCC 267 GCCTTTCTACAGTGGCCTTATCCCT 268CCTTTCTACAGTGGCCTTATCCCT 269 CTTTCTACAGTGGCCTTATCCCT 270TTTCTACAGTGGCCTTATCCCT 271 TTCTACAGTGGCCTTATCCCT 272TCTACAGTGGCCTTATCCCT 273 CTACAGTGGCCTTATCCCT 274 TACAGTGGCCTTATCCCT 275ACAGTGGCCTTATCCCT 276 CAGTGGCCTTATCCCT 277 AGTGGCCTTATCCCT

Examples of siRNA molecules targeting mRNAs transcribed from the DNAsequences listed in Table 2 are provided in Table 3 below.

TABLE 3 SEQ ID NO siRNA molecule sequence 278 GGCCUUGGUAUGUUCCUGCUUCAUG279 GCCUUGGUAUGUUCCUGCUUCAUG 280 CCUUGGUAUGUUCCUGCUUCAUG 281CUUGGUAUGUUCCUGCUUCAUG 282 UUGGUAUGUUCCUGCUUCAUG 283UGGUAUGUUCCUGCUUCAUG 284 GGUAUGUUCCUGCUUCAUG 285 GUAUGUUCCUGCUUCAUG 286UAUGUUCCUGCUUCAUG 287 AUGUUCCUGCUUCAUG 288 UGUUCCUGCUUCAUG 289GCCUUGGUAUGUUCCUGCUUCAUGC 290 CCUUGGUAUGUUCCUGCUUCAUGC 291CUUGGUAUGUUCCUGCUUCAUGC 292 UUGGUAUGUUCCUGCUUCAUGC 293UGGUAUGUUCCUGCUUCAUGC 294 GGUAUGUUCCUGCUUCAUGC 295 GUAUGUUCCUGCUUCAUGC296 UAUGUUCCUGCUUCAUGC 297 AUGUUCCUGCUUCAUGC 298 UGUUCCUGCUUCAUGC 299GUUCCUGCUUCAUGC 300 CCUUGGUAUGUUCCUGCUUCAUGCC 301CUUGGUAUGUUCCUGCUUCAUGCC 302 UUGGUAUGUUCCUGCUUCAUGCC 303UGGUAUGUUCCUGCUUCAUGCC 304 GGUAUGUUCCUGCUUCAUGCC 305GUAUGUUCCUGCUUCAUGCC 306 UAUGUUCCUGCUUCAUGCC 307 AUGUUCCUGCUUCAUGCC 308UGUUCCUGCUUCAUGCC 309 GUUCCUGCUUCAUGCC 310 UUCCUGCUUCAUGCC 311CUUGGUAUGUUCCUGCUUCAUGCCU 312 UUGGUAUGUUCCUGCUUCAUGCCU 313UGGUAUGUUCCUGCUUCAUGCCU 314 GGUAUGUUCCUGCUUCAUGCCU 315GUAUGUUCCUGCUUCAUGCCU 316 UAUGUUCCUGCUUCAUGCCU 317 AUGUUCCUGCUUCAUGCCU318 UGUUCCUGCUUCAUGCCU 319 GUUCCUGCUUCAUGCCU 320 UUCCUGCUUCAUGCCU 321UCCUGCUUCAUGCCU 322 UUGGUAUGUUCCUGCUUCAUGCCUU 323UGGUAUGUUCCUGCUUCAUGCCUU 324 GGUAUGUUCCUGCUUCAUGCCUU 325GUAUGUUCCUGCUUCAUGCCUU 326 UAUGUUCCUGCUUCAUGCCUU 327AUGUUCCUGCUUCAUGCCUU 328 UGUUCCUGCUUCAUGCCUU 329 GUUCCUGCUUCAUGCCUU 330UUCCUGCUUCAUGCCUU 331 UCCUGCUUCAUGCCUU 332 CCUGCUUCAUGCCUU 333UGGUAUGUUCCUGCUUCAUGCCUUU 334 GGUAUGUUCCUGCUUCAUGCCUUU 335GUAUGUUCCUGCUUCAUGCCUUU 336 UAUGUUCCUGCUUCAUGCCUUU 337AUGUUCCUGCUUCAUGCCUUU 338 UGUUCCUGCUUCAUGCCUUU 339 GUUCCUGCUUCAUGCCUUU340 UUCCUGCUUCAUGCCUUU 341 UCCUGCUUCAUGCCUUU 342 CCUGCUUCAUGCCUUU 343CUGCUUCAUGCCUUU 344 GGUAUGUUCCUGCUUCAUGCCUUUC 345GUAUGUUCCUGCUUCAUGCCUUUC 346 UAUGUUCCUGCUUCAUGCCUUUC 347AUGUUCCUGCUUCAUGCCUUUC 348 UGUUCCUGCUUCAUGCCUUUC 349GUUCCUGCUUCAUGCCUUUC 350 UUCCUGCUUCAUGCCUUUC 351 UCCUGCUUCAUGCCUUUC 352CCUGCUUCAUGCCUUUC 353 CUGCUUCAUGCCUUUC 354 UGCUUCAUGCCUUUC 355GUAUGUUCCUGCUUCAUGCCUUUCU 356 UAUGUUCCUGCUUCAUGCCUUUCU 357AUGUUCCUGCUUCAUGCCUUUCU 358 UGUUCCUGCUUCAUGCCUUUCU 359GUUCCUGCUUCAUGCCUUUCU 360 UUCCUGCUUCAUGCCUUUCU 361 UCCUGCUUCAUGCCUUUCU362 CCUGCUUCAUGCCUUUCU 363 CUGCUUCAUGCCUUUCU 364 UGCUUCAUGCCUUUCU 365GCUUCAUGCCUUUCU 366 UAUGUUCCUGCUUCAUGCCUUUCUA 367AUGUUCCUGCUUCAUGCCUUUCUA 368 UGUUCCUGCUUCAUGCCUUUCUA 369GUUCCUGCUUCAUGCCUUUCUA 370 UUCCUGCUUCAUGCCUUUCUA 371UCCUGCUUCAUGCCUUUCUA 372 CCUGCUUCAUGCCUUUCUA 373 CUGCUUCAUGCCUUUCUA 374UGCUUCAUGCCUUUCUA 375 GCUUCAUGCCUUUCUA 376 CUUCAUGCCUUUCUA 377AUGUUCCUGCUUCAUGCCUUUCUAC 378 UGUUCCUGCUUCAUGCCUUUCUAC 379GUUCCUGCUUCAUGCCUUUCUAC 380 UUCCUGCUUCAUGCCUUUCUAC 381UCCUGCUUCAUGCCUUUCUAC 382 CCUGCUUCAUGCCUUUCUAC 383 CUGCUUCAUGCCUUUCUAC384 UGCUUCAUGCCUUUCUAC 385 GCUUCAUGCCUUUCUAC 386 CUUCAUGCCUUUCUAC 387UUCAUGCCUUUCUAC 388 UGUUCCUGCUUCAUGCCUUUCUACA 389GUUCCUGCUUCAUGCCUUUCUACA 390 UUCCUGCUUCAUGCCUUUCUACA 391UCCUGCUUCAUGCCUUUCUACA 392 CCUGCUUCAUGCCUUUCUACA 393CUGCUUCAUGCCUUUCUACA 394 UGCUUCAUGCCUUUCUACA 395 GCUUCAUGCCUUUCUACA 396CUUCAUGCCUUUCUACA 397 UUCAUGCCUUUCUACA 398 UCAUGCCUUUCUACA 399GUUCCUGCUUCAUGCCUUUCUACAG 400 UUCCUGCUUCAUGCCUUUCUACAG 401UCCUGCUUCAUGCCUUUCUACAG 402 CCUGCUUCAUGCCUUUCUACAG 403CUGCUUCAUGCCUUUCUACAG 404 UGCUUCAUGCCUUUCUACAG 405 GCUUCAUGCCUUUCUACAG406 CUUCAUGCCUUUCUACAG 407 UUCAUGCCUUUCUACAG 408 UCAUGCCUUUCUACAG 409CAUGCCUUUCUACAG 410 UUCCUGCUUCAUGCCUUUCUACAGU 411UCCUGCUUCAUGCCUUUCUACAGU 412 CCUGCUUCAUGCCUUUCUACAGU 413CUGCUUCAUGCCUUUCUACAGU 414 UGCUUCAUGCCUUUCUACAGU 415GCUUCAUGCCUUUCUACAGU 416 CUUCAUGCCUUUCUACAGU 417 UUCAUGCCUUUCUACAGU 418UCAUGCCUUUCUACAGU 419 CAUGCCUUUCUACAGU 420 AUGCCUUUCUACAGU 421UCCUGCUUCAUGCCUUUCUACAGUG 422 CCUGCUUCAUGCCUUUCUACAGUG 423CUGCUUCAUGCCUUUCUACAGUG 424 UGCUUCAUGCCUUUCUACAGUG 425GCUUCAUGCCUUUCUACAGUG 426 CUUCAUGCCUUUCUACAGUG 427 UUCAUGCCUUUCUACAGUG428 UCAUGCCUUUCUACAGUG 429 CAUGCCUUUCUACAGUG 430 AUGCCUUUCUACAGUG 431UGCCUUUCUACAGUG 432 CCUGCUUCAUGCCUUUCUACAGUGG 433CUGCUUCAUGCCUUUCUACAGUGG 434 UGCUUCAUGCCUUUCUACAGUGG 435GCUUCAUGCCUUUCUACAGUGG 436 CUUCAUGCCUUUCUACAGUGG 437UUCAUGCCUUUCUACAGUGG 438 UCAUGCCUUUCUACAGUGG 439 CAUGCCUUUCUACAGUGG 440AUGCCUUUCUACAGUGG 441 UGCCUUUCUACAGUGG 442 GCCUUUCUACAGUGG 443CUGCUUCAUGCCUUUCUACAGUGGC 444 UGCUUCAUGCCUUUCUACAGUGGC 445GCUUCAUGCCUUUCUACAGUGGC 446 CUUCAUGCCUUUCUACAGUGGC 447UUCAUGCCUUUCUACAGUGGC 448 UCAUGCCUUUCUACAGUGGC 449 CAUGCCUUUCUACAGUGGC450 AUGCCUUUCUACAGUGGC 451 UGCCUUUCUACAGUGGC 452 GCCUUUCUACAGUGGC 453CCUUUCUACAGUGGC 454 UGCUUCAUGCCUUUCUACAGUGGCC 455GCUUCAUGCCUUUCUACAGUGGCC 456 CUUCAUGCCUUUCUACAGUGGCC 457UUCAUGCCUUUCUACAGUGGCC 458 UCAUGCCUUUCUACAGUGGCC 459CAUGCCUUUCUACAGUGGCC 460 AUGCCUUUCUACAGUGGCC 461 UGCCUUUCUACAGUGGCC 462GCCUUUCUACAGUGGCC 463 CCUUUCUACAGUGGCC 464 CUUUCUACAGUGGCC 465GCUUCAUGCCUUUCUACAGUGGCCU 466 CUUCAUGCCUUUCUACAGUGGCCU 467UUCAUGCCUUUCUACAGUGGCCU 468 UCAUGCCUUUCUACAGUGGCCU 469CAUGCCUUUCUACAGUGGCCU 470 AUGCCUUUCUACAGUGGCCU 471 UGCCUUUCUACAGUGGCCU472 GCCUUUCUACAGUGGCCU 473 CCUUUCUACAGUGGCCU 474 CUUUCUACAGUGGCCU 475UUUCUACAGUGGCCU 476 CUUCAUGCCUUUCUACAGUGGCCUU 477UUCAUGCCUUUCUACAGUGGCCUU 478 UCAUGCCUUUCUACAGUGGCCUU 479CAUGCCUUUCUACAGUGGCCUU 480 AUGCCUUUCUACAGUGGCCUU 481UGCCUUUCUACAGUGGCCUU 482 GCCUUUCUACAGUGGCCUU 483 CCUUUCUACAGUGGCCUU 484CUUUCUACAGUGGCCUU 485 UUUCUACAGUGGCCUU 486 UUCUACAGUGGCCUU 487UUCAUGCCUUUCUACAGUGGCCUUA 488 UCAUGCCUUUCUACAGUGGCCUUA 489CAUGCCUUUCUACAGUGGCCUUA 490 AUGCCUUUCUACAGUGGCCUUA 491UGCCUUUCUACAGUGGCCUUA 492 GCCUUUCUACAGUGGCCUUA 493 CCUUUCUACAGUGGCCUUA494 CUUUCUACAGUGGCCUUA 495 UUUCUACAGUGGCCUUA 496 UUCUACAGUGGCCUUA 497UCUACAGUGGCCUUA 498 UCAUGCCUUUCUACAGUGGCCUUAU 499CAUGCCUUUCUACAGUGGCCUUAU 500 AUGCCUUUCUACAGUGGCCUUAU 501UGCCUUUCUACAGUGGCCUUAU 502 GCCUUUCUACAGUGGCCUUAU 503CCUUUCUACAGUGGCCUUAU 504 CUUUCUACAGUGGCCUUAU 505 UUUCUACAGUGGCCUUAU 506UUCUACAGUGGCCUUAU 507 UCUACAGUGGCCUUAU 508 CUACAGUGGCCUUAU 509CAUGCCUUUCUACAGUGGCCUUAUC 510 AUGCCUUUCUACAGUGGCCUUAUC 511UGCCUUUCUACAGUGGCCUUAUC 512 GCCUUUCUACAGUGGCCUUAUC 513CCUUUCUACAGUGGCCUUAUC 514 CUUUCUACAGUGGCCUUAUC 515 UUUCUACAGUGGCCUUAUC516 UUCUACAGUGGCCUUAUC 517 UCUACAGUGGCCUUAUC 518 CUACAGUGGCCUUAUC 519UACAGUGGCCUUAUC 520 AUGCCUUUCUACAGUGGCCUUAUCC 521UGCCUUUCUACAGUGGCCUUAUCC 522 GCCUUUCUACAGUGGCCUUAUCC 523CCUUUCUACAGUGGCCUUAUCC 524 CUUUCUACAGUGGCCUUAUCC 525UUUCUACAGUGGCCUUAUCC 526 UUCUACAGUGGCCUUAUCC 527 UCUACAGUGGCCUUAUCC 528CUACAGUGGCCUUAUCC 529 UACAGUGGCCUUAUCC 530 ACAGUGGCCUUAUCC 531UGCCUUUCUACAGUGGCCUUAUCCC 532 GCCUUUCUACAGUGGCCUUAUCCC 533CCUUUCUACAGUGGCCUUAUCCC 534 CUUUCUACAGUGGCCUUAUCCC 535UUUCUACAGUGGCCUUAUCCC 536 UUCUACAGUGGCCUUAUCCC 537 UCUACAGUGGCCUUAUCCC538 CUACAGUGGCCUUAUCCC 539 UACAGUGGCCUUAUCCC 540 UCAGUGGCCUUAUCCC 541CAGUGGCCUUAUCCC 542 GCCUUUCUACAGUGGCCUUAUCCCU 543CCUUUCUACAGUGGCCUUAUCCCU 544 CUUUCUACAGUGGCCUUAUCCCU 545UUUCUACAGUGGCCUUAUCCCU 546 UUCUACAGUGGCCUUAUCCCU 547UCUACAGUGGCCUUAUCCCU 548 CUACAGUGGCCUUAUCCCU 549 UACAGUGGCCUUAUCCCU 550ACAGUGGCCUUAUCCCU 551 CAGUGGCCUUAUCCCU 552 AGUGGCCUUAUCCCU

A siRNA molecule may be capable of inhibiting the expression of a targetgene, such as the PNPLA3 148M allele. Herein, the terms “silencing” or“reducing” may be used interchangeably with “inhibiting.” To examine theextent of inhibition of expression by a siRNA, a siRNA of interest maybe added to a test sample and monitored for expression along with anegative control sample to which the siRNA was not added. Preferably, anegative control sample will be similar to the test sample. Morepreferably, the negative control sample will be identical to the testsample. Examples of negative control samples include untreated samples,samples to which a siRNA-free buffer was added, or samples to which anegative control or mock siRNA was added. Expression in the test samplecan then be compared to expression in the negative control sample.Expression may be measured by the detection of any expression productknown in the art or yet to be disclosed. Typical expression productsthat may be detected include RNA and protein.

Methods known in the art for the detection and quantification of RNAexpression in a sample include northern blotting and in situhybridization (Parker and Barnes, Methods in Molecular Biology 106,247-283 (1999) incorporated by reference herein in its entirety); RNAseprotection assays (Hod, Biotechniques 13, 852-854 (1992) incorporated byreference herein in its entirety); and PCR-based methods, such asreverse transcription polymerase chain reaction (RT-PCR) (Weis et al.,Trends in Genetics 8, 263-264 (1992) incorporated by reference herein inits entirety). Representative methods for sequencing-based geneexpression analysis include Serial Analysis of Gene Expression (SAGE),and gene expression analysis by massively parallel signature sequencing(MPSS). (See Mardis E R, Annu Rev Genomics Hum Genet 9, 387-402(2008))(the content of which is incorporated by reference herein in itsentirety).

Proteins, for example, can be detected and quantified through epitopesrecognized by polyclonal and/or monoclonal antibodies used in methodssuch as ELISA, immunoblot assays, flow cytometric assays,immunohistochemical assays, radioimmuno assays, Western blot assays, animmunofluorescent assays, chemiluminescent assays and other polypeptidedetection strategies. Proteins may also be detected by mass spectrometryassays (potentially coupled to immunoaffinity assays) includingmatrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)mass mapping and liquid chromatography/quadrupole time-of-flightelectrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS).Additionally, protein expression may be detected by tagging of proteinsseparated by two-dimensional polyacrylamide gel electrophoresis(2D-PAGE), (Kiernan et al, Anal Biochem 301, 49-56 (2002); Poutanen etal, Mass Spectrom 15, 1685-1692 (2001) the content of each of which isincorporated by reference herein in its entirety) or any other method ofdetecting protein.

In general, negative control samples are assigned a value of 100%.Inhibition of expression of a target gene may be achieved when theexpression of the test sample relative to the control sample is 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, 1%, less than 1% or 0%. Expression of a test samplerelative to a negative control sample may also be presented in terms offold reduction, such as a 2-fold, 3-fold, 5-fold, 10-fold, 20-fold,50-fold, or 100-fold less expression than the negative control sample.

Two or more nucleic acid sequences or subsequences may be referred to asbeing substantially identical, meaning that they are exactly the same orhave a specified percentage of nucleotides that are the same.Substantially identical nucleotides may have at least 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or more than 95% identity over a specifiedregion when compared and aligned for maximum correspondence. Thisdefinition, when the context indicates, also refers to the complement ofa sequence. Preferably, the substantial identity exists over a regionthat is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60nucleotides in length.

SiRNA molecules can be provided in several forms including, e.g., as oneor more isolated siRNA duplexes, as longer double-stranded RNA (dsRNA),or as siRNA or dsRNA transcribed from a transcriptional cassette in aDNA plasmid. The siRNA sequences may have overhangs (as 3′ or 5′overhangs as described in Elbashir et al, Genes Dev 15, 188 (2001) orNykanen et al, Cell 107, 309 (2001), the content of each of which isincorporated by reference herein in its entirety) or may lack overhangs(i.e., have blunt ends).

One or more DNA plasmids encoding one or more siRNA templates may beused to provide siRNA. siRNA can be transcribed as sequences thatautomatically fold into duplexes with hairpin loops from DNA templatesin plasmids having RNA polymerase Ill transcriptional units, forexample, based on the naturally occurring transcription units for smallnuclear RNA U6 or human RNase P RNA H1 (Brummelkamp et al, Science 296,550 (2002); Donze et al, Nucleic Acids Res 30, e46 (2002); Paddison etal, Genes Dev 16, 948 (2002); Yu et al, Proc Natl Acad Sci USA 99, 6047(2002); Lee et al, Nat Biotech, 20, 500 (2002); Miyagishi et al, NatBiotech 20, 497 (2002); Paul et al, Nat Biotech, 20, 505 (2002); and Suiet al, Proc Natl Acad Sci USA, 99, 5515 (2002); the content of each ofwhich is incorporated by reference herein in its entirety). Typically, atranscriptional unit or cassette will contain an RNA transcript promotersequence, such as an H1-RNA or a U6 promoter, operably linked to atemplate for transcription of a desired siRNA sequence and a terminationsequence, comprised of 2-3 uridine residues and a polythymidine (T5)sequence (polyadenylation signal) (Brummelkamp et al (2002) supra). Theselected promoter can provide for constitutive or inducibletranscription. Compositions and methods for DNA-directed transcriptionof RNA interference molecules are described in detail in U.S. Pat. No.6,573,099, incorporated by reference herein in its entirety. Thetranscriptional unit is incorporated into a plasmid or DNA vector fromwhich the interfering RNA is transcribed. Plasmids suitable for in vivodelivery of genetic material for therapeutic purposes are described indetail in U.S. Pat. Nos. 5,962,428 and 5,910,488, the content of each ofwhich is incorporated by reference herein in its entirety. The selectedplasmid can provide for transient or stable delivery of a nucleic acidto a target cell. It will be apparent to those of skill in the art thatplasmids originally designed to express desired gene sequences can bemodified to contain a transcriptional unit cassette for transcription ofsiRNA.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene 25, 263-269 (1983);Sambrook and Russell, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor N.Y., (2001), thecontent of each of which is incorporated by reference herein in itsentirety) as are PCR methods (see, U.S. Pat. Nos. 4,683,195 and4,683,202; PCR Protocols: A Guide to Methods and Applications, Innis etal, eds, (1990), the content of each of which is incorporated byreference herein in its entirety). Expression libraries are also wellknown to those of skill in the art. Additional basic texts disclosingthe general methods of use in this invention include Sambrook andRussell (2001) supra; Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994), the content of each of which isincorporated by reference herein in its entirety.

A siRNA molecule may be chemically synthesized. In one example ofchemical synthesis, a single-stranded nucleic acid that includes thesiRNA duplex sequence can be synthesized using any of a variety oftechniques known in the art, such as those described in Usman et al, JAm Chem Soc, 109, 7845 (1987); Scaringe et al, Nucl Acids Res, 18, 5433(1990); Wincott et al, Nucl Acids Res, 23, 2677-2684 (1995); and Wincottet al, Methods Mol Bio 74, 59 (1997), the content of each of which isincorporated by reference herein in its entirety. Synthesis of thesingle-stranded nucleic acid makes use of common nucleic acid protectingand coupling groups, such as dimethoxytrityl at the 5′-end andphosphoramidites at the 3′-end. As a non-limiting example, small scalesyntheses can be conducted on an Applied Biosystems synthesizer (ThermoFisher Scientific, Waltham, Mass.) using a 0.2 micromolar scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides.Alternatively, syntheses at the 0.2 micromolar scale can be performed ona 96-well plate synthesizer from Thermo Fisher Scientific. However,larger or smaller scale synthesis are also encompassed by the invention,including any method of synthesis now known or yet to be disclosed.Suitable reagents for synthesis of siRNA single-stranded molecules,methods for RNA deprotection, and methods for RNA purification are knownto those of skill in the art.

In certain embodiments, siRNA can be synthesized via a tandem synthesistechnique, wherein both strands are synthesized as a single continuousfragment or strand separated by a linker that is subsequently cleaved toprovide separate fragments or strands that hybridize to form a siRNAduplex. Linkers may be any linker, including a polynucleotide linker ora non-nucleotide linker. The tandem synthesis of siRNA can be readilyadapted to both multiwell/multiplate synthesis platforms as well aslarge scale synthesis platforms employing batch reactors, synthesiscolumns, and the like. In some embodiments, siRNA can be assembled fromtwo distinct single-stranded molecules, wherein one strand includes thesense strand and the other includes the antisense strand of the siRNA.For example, each strand can be synthesized separately and joinedtogether by hybridization or ligation following synthesis and/ordeprotection. Either the sense or the antisense strand may containadditional nucleotides that are not complementary to one another and donot form a double stranded siRNA. In certain instances, siRNA moleculescan be synthesized as a single continuous fragment, where theself-complementary sense and antisense regions hybridize to form a siRNAduplex having hairpin secondary structure.

A siRNA molecule may comprise a duplex having two complementary strandsthat form a double-stranded region with least one modified nucleotide inthe double-stranded region. The modified nucleotide may be on one strandor both. If the modified nucleotide is present on both strands, it maybe in the same or different positions on each strand. A modified siRNAmay be less immunostimulatory than a corresponding unmodified siRNAsequence, but retains the capability of silencing the expression of atarget sequence.

Examples of modified nucleotides suitable for use in the presentinvention include, but are not limited to, ribonucleotides having a2′-O-methyl (2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl,2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group.Modified nucleotides having a conformation such as those described inthe art, for example in Saenger, Principles of Nucleic Acid Structure,Springer-Verlag Ed. (1984), incorporated by reference herein in itsentirety, are also suitable for use in siRNA molecules. Other modifiednucleotides include, without limitation: locked nucleic acid (LNA)nucleotides, G-clamp nucleotides, or nucleotide base analogs. LNAnucleotides include but need not be limited to 2′-O,4′-C-methylene-(D-ribofuranosyl)nucleotides), 2′-O-(2-methoxyethyl)(MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy-2′-chloro (2Cl) nucleotides, and 2′-azidonucleotides. A G-clamp nucleotide refers to a modified cytosine analogwherein the modifications confer the ability to hydrogen bond bothWatson-Crick and Hoogsteen faces of a complementary guanine nucleotidewithin a duplex (Lin et al, J Am Chem Soc, 120, 8531-8532 (1998)incorporated by reference herein in its entirety). Nucleotide baseanalogs include for example, C-phenyl, C-naphthyl, other aromaticderivatives, inosine, azole carboxamides, and nitroazole derivativessuch as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole(Loakes, Nucl Acids Res, 29, 2437-2447 (2001) incorporated by referenceherein in its entirety).

A siRNA molecule may comprise one or more chemical modifications such asterminal cap moieties, phosphate backbone modifications, and the like.Examples of classes of terminal cap moieties include, withoutlimitation, inverted deoxy abasic residues, glyceryl modifications,4′,5′-methylene nucleotides, 1-(.beta.-D-erythrofuranosyl) nucleotides,4′-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitolnucleotides, L-nucleotides, .alpha.-nucleotides, modified basenucleotides, threo pentofuranosyl nucleotides, acyclic 3′,4′-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic3,5-dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties,3′-3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties,3′-2′-inverted abasic moieties, 5′-5′-inverted nucleotide moieties,5′-5′-inverted abasic moieties, 3′-5′-inverted deoxy abasic moieties,5′-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3 aminopropylphosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate,hydroxypropyl phosphate, 1,4-butanediol phosphate, 3′-phosphoramidate,5′ phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate,and bridging or non-bridging methylphosphonate or 5′-mercapto moieties(see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al, Tetrahedron 49,1925 (1993); the content of each of which is incorporated by referenceherein in its entirety). Non-limiting examples of phosphate backbonemodifications (i.e., resulting in modified internucleotide linkages)include phosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate, carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker et al,Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al,Antisense Research, ACS, 24-39 (1994); the content of each of which isincorporated by reference herein in its entirety). Such chemicalmodifications can occur at the 5′-end and/or 3′-end of the sense strand,antisense strand, or both strands of the siRNA.

The sense and/or antisense strand of a siRNA may comprise a 3′-terminaloverhang having 1 to 4 or more 2′-deoxyribonucleotides and/or anycombination of modified and unmodified nucleotides. Additional examplesof modified nucleotides and types of chemical modifications that can beintroduced into the modified siRNA molecules of the present inventionare described, e.g., in UK Patent No. GB 2,397,818 B and U.S. PatentPublication Nos. 20040192626 and 20050282188, the content of each ofwhich is incorporated by reference herein in its entirety.

A siRNA molecule may comprise one or more non-nucleotides in one or bothstrands of the siRNA. A non-nucleotide may be any subunit, functionalgroup, or other molecular entity capable of being incorporated into anucleic acid chain in the place of one or more nucleotide units that isnot or does not comprise a commonly recognized nucleotide base such asadenosine, guanine, cytosine, uracil, or thymine, such as a sugar orphosphate.

Chemical modification of siRNA may comprise attaching a conjugate to asiRNA molecule. The conjugate can be attached at the 5′- and/or the3′-end of the sense and/or the antisense strand of the siRNA via acovalent attachment such as a nucleic acid or non-nucleic acid linker.The conjugate can be attached to the siRNA through a carbamate group orother linking group (see, e.g., U.S. Patent Publication Nos.20050074771, 20050043219, and 20050158727, the content of each of whichis incorporated by reference herein in its entirety). A conjugate may beadded to siRNA for any of a number of purposes. For example, theconjugate may be a molecular entity that facilitates the delivery ofsiRNA into a cell or may be a molecule that comprises a drug or label.Examples of conjugate molecules suitable for attachment to siRNA of thepresent invention include, without limitation, steroids such ascholesterol, glycols such as polyethylene glycol (PEG), human serumalbumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates(e.g., folic acid, folate analogs and derivatives thereof), sugars(e.g., galactose, galactosamine, N-acetyl galactosamine, glucose,mannose, fructose, fucose, etc.), phospholipids, peptides, ligands forcellular receptors capable of mediating cellular uptake, andcombinations thereof (see, e.g., U.S. Patent Publication Nos.20030130186, 20040110296, and 20040249178; U.S. Pat. No. 6,753,423; thecontent of each of which is incorporated by reference herein in itsentirety). Other examples include the lipophilic moiety, vitamin,polymer, peptide, protein, nucleic acid, small molecule,oligosaccharide, carbohydrate cluster, intercalator, minor groovebinder, cleaving agent, and cross-linking agent conjugate moleculesdescribed in U.S. Patent Publication Nos. 20050119470 and 20050107325,the content of each of which is incorporated by reference herein in itsentirety. Other examples include the 2′-O-alkyl amine, 2′-O-alkoxyalkylamine, polyamine, C5-cationic modified pyrimidine, cationic peptide,guanidinium group, amidininium group, cationic amino acid conjugatemolecules described in U.S. Patent Publication No. 20050153337,incorporated by reference herein in its entirety. Additional examples ofconjugate molecules include a hydrophobic group, a membrane activecompound, a cell penetrating compound, a cell targeting signal, aninteraction modifier, or a steric stabilizer as described in U.S. PatentPublication No. 20040167090, incorporated by reference herein in itsentirety. Further examples include the conjugate molecules described inU.S. Patent Publication No. 20050239739, incorporated by referenceherein in its entirety.

The type of conjugate used and the extent of conjugation to the siRNAcan be evaluated for improved pharmacokinetic profiles, bioavailability,and/or stability of the siRNA while retaining activity. As such, oneskilled in the art can screen siRNA molecules having various conjugatesattached thereto to identify siRNA conjugates having improved propertiesusing any of a variety of well-known in vitro cell culture or in vivoanimal models including the negative-controlled expression studiesdescribed above.

A siRNA may be incorporated into carrier systems containing siRNAmolecules described herein. The carrier system may be a lipid-basedcarrier system such as a stabilized nucleic acid-lipid particle (e.g.,SNALP or SPLP), cationic lipid or liposome nucleic acid complexes (i.e.,lipoplexes), a liposome, a micelle, a virosome, or a mixture thereof. Inother embodiments, the carrier system may be a polymer-based carriersystem such as a cationic polymer-nucleic acid complex (i.e., polyplex).In additional embodiments, the carrier system can be acyclodextrin-based carrier system such as a cyclodextrin polymer-nucleicacid complex (see US Patent Application Publication 20070218122,incorporated by reference herein in its entirety). In furtherembodiments, the carrier system may be a protein-based carrier systemsuch as a cationic peptide-nucleic acid complex. A siRNA molecule mayalso be delivered as naked siRNA.

In certain embodiments, the carrier system can be a nanoparticle thatincludes low molecular weight polyethyleneimine (LPEI) or itsderivatives (e.g., disulfide crosslinked polyethyleneimine (CLPEI)) anda lipid. The lipid may be a bile acid, such as cholic acid, deoxycholicacid, and lithocholic acid. Such carrier systems are described furtherin the Examples below. Other exemplary carrier systems are described forexample in Wittrup et al. (Nature Reviews/Genetics, 16:543-552, 2015),the content of which is incorporated by reference herein in itsentirety.

The compositions of the invention are particularly useful for treating asubject (e.g., a mammalian subject, e.g., human, child or adult) with achronic liver disease or alcoholic liver disease (ALD). Chronic liverdisease refers to diseases of the liver that last over a period of sixmonths. It includes of a wide range of liver pathologies which includeinflammation (chronic hepatitis), liver cirrhosis, and hepatocellularcarcinoma. Alcoholic liver disease (ALD) typically occurs after years ofheavy drinking. Over time, scarring and cirrhosis can occur. Cirrhosisis the final phase of alcoholic liver disease. There may be no symptoms,or symptoms may come on slowly, depending on how well the liver isworking. Symptoms tend to be worse after a period of heavy drinking.Early symptoms include: fatigue and loss of energy; poor appetite andweight loss; nausea or belly pain; small, or red spider-like bloodvessels on the skin As liver function worsens, symptoms may include:fluid buildup of the legs (edema) and in the abdomen (ascites); yellowcolor in the skin, mucous membranes, or eyes (jaundice); redness on thepalms of the hands; easy bruising and abnormal bleeding; confusion orproblems thinking; or pale or clay-colored stools. In men, symptoms mayalso include impotence, shrinking of the testicles, and breast swelling.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systematically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

When the compounds of the present invention are administered aspharmaceuticals, to humans and mammals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient, (e.g., a siRNA orantisense oligonucleotide of the invention) and/or derivative thereof,in combination with a pharmaceutically acceptable carrier.

The effective dosage of each agent can readily be determined by theskilled person, having regard to typical factors each as the age,weight, sex and clinical history of the patient. In general, a suitabledaily dose of a compound of the invention will be that amount of thecompound which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. Generally, doses of the compounds of this invention fora patient, when used for the indicated effects; will range from about0.1 mg to about 250 mg per kilogram of body weight per day, morepreferably from about 1 mg to about 60 mg per kg per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The pharmaceutical compositions of the invention include a“therapeutically effective amount” or a “prophylactically effectiveamount” of one or more of the compounds of the present invention, orfunctional derivatives thereof. An “effective amount” is the amount asdefined herein in the definition section and refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result, e.g., a diminishment or prevention ofeffects associated with neuropathic and/or inflammatory pain. Atherapeutically effective amount of a compound of the present inventionor functional derivatives thereof may vary according to factors such asthe disease state, age, sex, and weight of the subject, and the abilityof the therapeutic compound to elicit a desired response in the subject.A therapeutically effective amount is also one in which any toxic ordetrimental effects of the therapeutic agent are outweighed by thetherapeutically beneficial effects.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to, or at an earlier stage of disease, theprophylactically effective amount may be less than the therapeuticallyeffective amount. A prophylactically or therapeutically effective amountis also one in which any toxic or detrimental effects of the compoundare outweighed by the beneficial effects.

Dosage regimens may be adjusted to provide the optimum desired response(e.g. a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigency of the therapeutic situation. It is especiallyadvantageous to formulate parenteral compositions in dosage unit formfor ease of administration and uniformity of dosage. Actual dosagelevels of the active ingredients in the pharmaceutical compositions ofthis invention may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular subject, composition, and mode ofadministration, without being toxic to the patient.

The term “dosage unit” as used herein refers to physically discreteunits suited as unitary dosages for the mammalian subjects to betreated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the compound, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

In some embodiments, therapeutically effective amount can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in other subjects. Generally, the therapeuticallyeffective amount is sufficient to reduce or inhibit neuropathic and/orinflammatory pain in a subject. In some embodiments, the therapeuticallyeffective amount is sufficient to eliminate neuropathic and/orinflammatory pain in a subject. Dosages for a particular patient can bedetermined by one of ordinary skill in the art using conventionalconsiderations, (e.g. by means of an appropriate, conventionalpharmacological protocol). A physician may, for example, prescribe arelatively low dose at first, subsequently increasing the dose until anappropriate response is obtained. The dose administered to a patient issufficient to effect a beneficial therapeutic response in the patientover time, or, e.g., to reduce symptoms, or other appropriate activity,depending on the application. The dose is determined by the efficacy ofthe particular formulation, and the activity, stability or serumhalf-life of the compounds of the invention or functional derivativesthereof, and the condition of the patient, as well as the body weight orsurface area of the patient to be treated. The size of the dose is alsodetermined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular vector,formulation, or the like in a particular subject. Therapeuticcompositions comprising one or more compounds of the invention orfunctional derivatives thereof are optionally tested in one or moreappropriate in vitro and/or in vivo animal models of disease, such asmodels of neuropathic and/or inflammatory pain, to confirm efficacy,tissue metabolism, and to estimate dosages, according to methods wellknown in the art. In particular, dosages can be initially determined byactivity, stability or other suitable measures of treatment vs.non-treatment (e.g., comparison of treated vs. untreated cells or animalmodels), in a relevant assay. Formulations are administered at a ratedetermined by the LD50 of the relevant formulation, and/or observationof any side-effects of compounds of the invention or functionalderivatives thereof at various concentrations, e.g., as applied to themass and overall health of the patient. Administration can beaccomplished via single or divided doses.

Administering typically involves administering pharmaceuticallyacceptable dosage forms, which means dosage forms of compounds describedherein, and includes, for example, tablets, dragees, powders, elixirs,syrups, liquid preparations, including suspensions, sprays, inhalantstablets, lozenges, emulsions, solutions, granules, capsules, andsuppositories, as well as liquid preparations for injections, includingliposome preparations. Techniques and formulations generally may befound in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa., latest edition, which is hereby incorporated by referencein its entirety. Administering may be carried out orally, intradermally,intramuscularly, intraperitoneally, intravenously, subcutaneously, orintranasally. Compounds may be administered alone or with suitablepharmaceutical carriers, and can be in solid or liquid form, such astablets, capsules, powders, solutions, suspensions, or emulsions.

A pharmaceutical composition containing the active ingredient may be ina form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from sweeteningagents, flavoring agents, coloring agents and preserving agents, inorder to provide pharmaceutically elegant and palatable preparations.Tablets contain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated by the techniques described in U.S. Pat. Nos. 4,256,108,4,166,452 and 4,265,874 (the content of each of which is incorporated byreference herein in its entirety), to form osmotic therapeutic tabletsfor control release.

Formulations for oral use may also be presented as hard gelatin capsulesin which the active ingredient is mixed with an inert solid diluent, forexample calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Formulations may also include complexes of the parent (unionized)compounds with derivatives of β-cyclodextrin, especiallyhydroxypropyl-β-cyclodextrin.

An alternative oral formulation can be achieved using acontrolled-release formulation, where the compound is encapsulated in anenteric coating.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents such as a naturally occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such a polyoxyethylene with partial esters derived from fattyacids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate. The aqueous suspensions may also contain one or morepreservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one ormore coloring agents, one or more flavoring agents, and one or moresweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified, for example sweetening, flavoring andcoloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally occurring phosphatides, for example soya bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate and condensation products ofthe said partial esters with ethylene oxide, for example polyoxyethylenesorbitan monooleate. The emulsions may also contain sweetening andflavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents. The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be in a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

Each active agent may also be administered in the form of suppositoriesfor rectal administration of the drug. These compositions can beprepared by mixing the drug with a suitable non-irritating excipientwhich is solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum to release the drug.Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, jellies, solutions or suspensionsare suitable. Topical application includes the use of mouth washes andgargles.

The term “pharmaceutical composition” means a composition comprising acompound as described herein and at least one component comprisingpharmaceutically acceptable carriers, diluents, adjuvants, excipients,or vehicles, such as preserving agents, fillers, disintegrating agents,wetting agents, emulsifying agents, suspending agents, sweeteningagents, flavoring agents, perfuming agents, antibacterial agents,antifungal agents, lubricating agents and dispensing agents, dependingon the nature of the mode of administration and dosage forms. The term“pharmaceutically acceptable carrier” is used to mean any carrier,diluent, adjuvant, excipient, or vehicle, as described herein. Examplesof suspending agents include ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample sugars, sodium chloride, and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monosterate andgelatin. Examples of suitable carriers, diluents, solvents, or vehiclesinclude water, ethanol, polyols, suitable mixtures thereof, vegetableoils (such as olive oil), and injectable organic esters such as ethyloleate. Examples of excipients include lactose, milk sugar, sodiumcitrate, calcium carbonate, and dicalcium phosphate. Examples ofdisintegrating agents include starch, alginic acids, and certain complexsilicates. Examples of lubricants include magnesium stearate, sodiumlauryl sulphate, talc, as well as high molecular weight polyethyleneglycols.

The term “pharmaceutically acceptable” means it is, within the scope ofsound medical judgment, suitable for use in contact with the cells ofhumans and lower animals without undue toxicity, irritation, allergicresponse, and the like, and are commensurate with a reasonablebenefit/risk ratio.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES

The PNPLA3 gene rs738409 C>G polymorphism is associated with severaltypes of liver disease (Shen, Journal of Lipid Research, 56:167-175,2015), the content of which is incorporated by reference herein in itsentirety. The G allele is associated with a significantly increased riskof chronic liver disease versus the C allele. Both the GC and GGgenotypes are associated with a significantly increased risk of chronicliver disease versus the CC genotype.

Accordingly, the I148M mutation (rs738409 C>G) in the PNPLA3 gene is astrong genetic risk factor for a series of chronic liver diseases,including nonalcoholic/alcoholic fatty liver disease, steatohepatitis,cirrhosis and hepatocellular carcinoma. It has been demonstrated thatoverexpression of the 148M isoform and not 148I is an important step forthe manifestation of these phenotypes. Meanwhile, 148M has beendemonstrated as a loss-of-function mutation that reduces the enzymaticactivity (triglycerides hydrolase and other unknown function) ascompared to the 148I isoform. This indicates that 148M plays apathogenic role in liver disease etiology in a dominant-negative manner.

Without being limited by any particular theory of mechanism of action,it is believed that reducing the expression of the 148M isoform willlead to reversing disease progress from simple steatosis,steatohepatitis, cirrhosis and even liver cancer. Accordingly, theinvention provides siRNA specifically targeting the 148M isoform toreduce its mRNA and protein expression. It has been found that thecompositions of the invention have minimal effect on the wild-type 148Iisoform. The data herein show that cell lines based model has confirmedthat reducing the 148M isoform leads to reduced fat accumulation inhuman HepG2 cell line.

Example 1: In Vitro Systems for Pharmacological Testing of siRNAMolecules

The present invention identifies a potent siRNA specificallydownregulating PNPLA3 148M isoform, namely 148MSi (SEQ ID NO.: 1) andassociated sequences SEQ ID NOs: 2 and 278-552. The therapeuticpotential of 148MSi may be further characterized through additionaltesting. To this end: 1) stable cell lines can be established constantlyexpressing PNPLA3148M-Luc and PNPLA3148I-Luc, which facilitates asubsequent high throughput optimization of 148MSi and its modifiedanalogs in targeting PNPLA3 148M; and 2) The therapeutic effectivenessof 148MSi can be examined in vitro, which can collect keypharmacological parameters to further foster the basis of 148MSi as atherapeutic agent.

To establish stable cell lines and perform a high throughput screeningfor more candidate siRNA targeting PNPLA3 148M, lentivirus particles maybe generated packing the vectors with each of the PNPLA3 148M-Luc andPNPLA3 148I-Luc fusion genes. The virus particles may be transduced intoHEK293 cells. Cells constantly expressing the fusion reporter proteinscan be selected using puromycin. Following the establishment of thesestable cells, a high-throughput screening can be performed to furtheridentify optimal siRNA candidates. Briefly, a series of siRNAsspecifically targeting the 148M allele can be designed by altering theirlength and/or position to target, as well as by artificially introducingmutations into the siRNA sequence. These siRNAs may be transientlytransfected into the stable cells to test their specificity and potencyby comparing to that of the 148MSi.

It may be that the stable cells are able to serve as an in vitro modelfor rapid screening of siRNA candidates, which may lead to theidentification of other siRNAs possessing equivalent to or even bettertherapeutic properties than 148MSi. Vectors may be created in the laband transient transfection may be performed as below.

To examine the therapeutic effectiveness of 148MSi in vitro, the highspecificity and potency of 148MSi may be demonstrated, as well as thepotential of 148MSi treatment in reducing triglycerides (TG)accumulation (a hallmark of hepatic steatosis) under glucose inductionof endogenous PNPLA3 148M in Huh-7 cells (See Examples below). However,glucose treatment may alter many pathways which might confound theeffect of PNPLA3. Artificially increasing the PNPLA3 148M expressionusing adenoviral vectors without integrating into the genome would beuseful to examine the focused effect of PNPLA3 148M on the induction ofhepatic steatosis. PNPLA3 148M and PNPLA3 148I can be cloned intoadenoviral vectors. PNPLA3 148M and PNPLA3 148I adenovirus particles canbe generated to transduce the Huh-7 cells, respectively. 148MSi, in aseries of concentrations, may be transfected into the cells usingLipofectamine 2000 (Thermal Fisher). The transfected cells can then betreated with free fatty acids (palmitic acid+oleic acid) for 48 hours,and TG accumulation may be measured using Oil Red O staining, and can befurther compared between the cells transduced with different PNPLA3isoforms.

Overexpression of PNPLA3148M isoform may significantly induce TGaccumulation as compared to the 148I isoform. Transfection of 148MSi cansignificantly reduce TG accumulation in a dose-dependent manner andspecifically in cells transduced with PNPLA3 148M but not PNPLA3 148I.Although Huh-7 is a PNPLA3 148M homozygote, the expression level ofPNPLA3 may be insufficient to induce TG accumulation. Previous studieshave shown that overexpression of PNPLA3 148M in hepatocyte indeedincreased TG accumulation, and knockdown of PNPLA3 using genericcommercial siRNAs can reverse that effect. 148MSi of the invention canoutperform generic siRNAs in PNPLA3 148M targeting as shown below andthe effect can likely be recapitulated in Huh-7. In one embodiment,short hairpin RNAs (shRNA) for 148MSi can be created and furtherpackaged into adenovirus to perform co-transfection with PNPLA3adenovirus.

Example 2: siRNA Targeting and Therapeutic Response In Vivo

To validate the cell-based data, useful mouse models can be created forin vivo evaluation of 148MSi. It has been reported that a short-termoverexpression of PNPLA3 148M variant using an adenoviral vector leadsto ˜3 fold increase in hepatic triglycerides. Transgenic mice thatoverexpress the PNPLA3 148M variant specifically in the liver alsocauses hepatic steatosis and dysregulated hepatic lipid metabolism.Thus, it is believed that overexpression of PNPLA3 148M variant in mouseliver should provide a useful tool for preclinical testing of theefficacy, pharmacodynamics, and pharmacokinetics.

Since adenoviral and lentiviral vectors can efficiently deliver geneproducts to the liver, their respective features (adenoviral vector doesnot integrate to the host genome but lentiviral vector does) can beadvantageous to the development of both acute and chronic mouse modelsexpressing human PNPLA3 148M (FIG. 7). DNA constructs may be created forboth systems, and large preparations can be made for animal injections.In order to validate the two models, PNPLA3 148I or PNPLA3 I148Madenoviruses (2×10⁹ pfu/mouse) or lentiviruses (5×10⁸ pfu/mouse) can beinjected. For the adenoviral PNPLA3 model, mice may be fasted for 4hours and sacrificed for blood and liver tissue collection 3 days afterthe injections. For the lentiviral PNPLA3 model, mice can be sacrificed1 month after the injections. Serum and hepatic triglycerides and freefatty acids can be analyzed using commercial assay kits (Dako). Hepaticlipid droplets may be analyzed by H&E and Oil Red 0 staining of liversections. In order to facilitate longitudinal monitoring of the PNPLA3expression, the two lentiviral vectors that carry PNPLA3 148I-Luc orPNPLA3 148M-Luc fusion gene mentioned in Example 1 above can be used.

Both adenoviral and lentiviral models for PNPLA3148M should develophepatic steatosis manifested by increased liver triglycerides and lipiddroplets. As an alternative strategy, an adeno-associated viral (AAV)vector system may be considered.

For the early phase of in vivo test, the adenoviral model can be used toassess the efficacy of PNPLA3 148M siRNAs. Two days after the adenoviralinjection, 148MSi can be delivered using the Invivofectamine 3.0 reagent(Thermo Fisher, 1.5 mg/kg) into mice via tail vein injection. Two dayslater, animals can be sacrificed for blood and liver tissue collection.PNPLA3 knockdown efficiency may be analyzed by qPCR. Serum and liver TGcan be analyzed as described above. To examine the long-term efficacy,the following lentiviral models may be used: PNPLA3 148I, PNPLA3 I148M,PNPLA3 148I-Luc and PNPLA3 148M-Luc. Two regimens can be performed onthese 4 models: 1) Injection of 148MSi+Invivofectamine 3.0 (1.5 mg/kg)two days after the lentiviral injection and then weekly injection at adose of 0.5 mg/kg for 1 month; 2) Injection of 148MSi+Invivofectamine3.0 4 weeks after the lentiviral injection and then weekly injection ata dose of 0.5 mg/kg for 1 month. The luciferase signal of each mouse canbe monitored weekly using, for example the Berthold LB981 NightOwlsystem (Berthold Technologies GmbH & Co. KG, Germany). For assessing thepotential toxicity, we can inject the same doses of siRNAs into wildtypeC57BL6/J mice using the same regimens as mentioned above. The mice bodyweight can be monitored every two days for 1 month. At the end, animalsmay be euthanized for gross toxicity examination. Liver enzymes (ALT andAST) can be measured and liver histology can be examined.

These animal models should provide useful tools for siRNA tests.According to cellular data, 148MSi should knock down the PNPLA3 148Mexpression and reverse hepatic steatosis. Although serious toxicity fromsiRNA/Invivofectamine particles is not expected, any side effect may benoted and minimized by adjusting the dose regimens.

Example 3: Nanoparticle Vectors for siRNA Delivery to the Liver

Delivery of siRNA requires a carrier system that can protect siRNA fromenzymatic degradation during circulation, prevent side effects due tonon-specific distribution in off-target tissues, and help the siRNA toenter target cells. A ternary gene complex called DPH complex has beenreported that includes nucleic acid, polycation (LPEI ordisulfide-crosslinked polyethyleneimine, CLPEI), and polysaccharide.That complex has achieved superior gene transfection efficiency to thatof commercial gene carriers like Lipofectamine or polyethyleneimine.However, due to its electrostatic nature of the complex, DPH is unstablein circulation and shows suboptimal gene transfection in vivo. It isbelieved that DPH with greater stability and smaller size can beproduced by grafting lipid components to the polycation component suchas LPEI (FIG. 2). That belief is supported by the fact that cholesterolgrafting reduces the size of polymeric micelle gene carriers from >500nm to <200 nm by stabilizing the core-shell structure and increasescolloidal stability. The lipid modification also facilitates cellularuptake of polyplexes, thereby increasing the gene transfectionefficiency. Making a lipid-modified LPEI should help form small (<100nm) gene carriers, which can stably circulate, effectively reach theliver, and transfect hepatocytes.

Bile acids such as cholic acid, deoxycholic acid, and lithocholic acid(LCA), or their derivatives can be used given their biocompatibility,commercial availability, and chemical reactivity. For preparation oflipid-grafted LPEI, bile acids may be grafted as a NHS-activated bileacid to the secondary amine of LPEI (FIG. 2A). A LCA-grafted linear PEI(LPEI), which can be readily translated to conjugates of LCA and LPEIderivatives such as CLPEI, has also been synthesized. It has beenconfirmed that a ternary complex containing LCA-grafted LPEI (LCA-LPEI)performed better than that with LPEI in reporter gene transfection. Thelipid content may be optimized considering its effect on particlestabilization and water solubility. It may be necessary to include asmall quantity of water-miscible organic solvent such as DMSO to enhancethe solubilization of the components and complex formation. Once ananoparticulate gene carrier is formed, it is possible to remove thesolvent via centrifugal filtration or dialysis. It is possible that theincreased stability may interfere with intracellular dissociation ofcomplex. Should this be the case, a lipid may be grafted to thesecondary amine via disulfide bond, which can facilitate intracellulardissociation of the lipid. An amine derivative of bile acid may first bereacted with dithiobis [succinimidyl propionate] and then reacted withLPEI in the presence of N,N-diisopropylethylamine. Alternatively,LCA-LPEI can be produced using carbonyldiimidazole (CDA) as a couplingagent. Structures of synthetic intermediates and purified products canbe confirmed by 1H-NMR analysis, based on the additional proton shiftsindicating the steroid framework in 0.6-1.8 ppm (FIG. 2B).

Ternary complexes of siRNA, LCA-LPEI, and polysaccharides (hyaluronicacid (HA) or dermatan sulfate (DS)) may be formed following methods asshown in Xu, P., Quick, G., Yeo, Y, (2009), Gene delivery through theuse of a hyaluronate-associated intracellularly degradable cross-linkedpolyethyleneimine, Biomaterials, 30(29):5834-5843, incorporated hereinby reference in its entirety. Briefly, an siRNA-polymer binary complexmay be first formed and then incubated with HA or DS to make a ternarycomplex. The transfection efficiency of 148Si can be tested in HepG2cells with the PNPLA3-Luc as a reporter system. The ternary complexesmay be characterized with respect to size and surface charge usingZetasizer Nano-ZS90. To examine particle size and its change in serum,the complexes can be incubated in 50% serum solution and sampled atdifferent time points. To estimate chemical stability duringcirculation, the complexes may be incubated in the presence of serum,nucleases, and heparin (representing anionic glycosaminoglycans). Theintegrity of ternary complex can be tested with agarose gelelectrophoresis as done in previous studies. See, Xu, P., et al., 2009,Gene delivery through the use of a hyaluronate-associatedintracellularly degradable crosslinked polyethyleneimine, Biomaterials30, 5834-5843 and Feng, M., et al., 2014, Stabilization of ahyaluronate-associated gene delivery system using calcium ions,Biomaterials Science, the content of each of which is incorporated byreference herein in its entirety. A complex that does not leach outsiRNA upon the challenges may be considered stable. Cellular uptake ofthe siRNA/LCA-LPEI/HA (or DS) ternary complex can be evaluated withconfocal microscopy and flow cytometry using fluorescently labeled siRNAand HepG2 cells. Uptake mechanism may be determined using inhibitors ofdifferent endocytosis pathways in each condition and changes in cellularuptake of complexes may be quantified with flow cytometry, andintracellular destination can be identified by co-localizing organellemarkers with complexes under a confocal microscope. Cytotoxicity of thecomplexes may be assessed by incubating HepG2 cells with gene complexesequivalent to 1 to 100 μg/mL CLPEI with a MTT assay and comparing withcells treated with PBS, 148MSi, or LgCLPEI.

Derivatives of cholesterol and LCA have been synthesized. A LCA-graftedlinear PEI (LPEI), which can be readily translated to LCA-CLPEIconjugates, has also been synthesized and it has been confirmed that aternary complex containing LCA-grafted LPEI performed better than thatwith LPEI in reporter gene transfection. The lipid content may beoptimized considering its effect on particle stabilization and watersolubility. It may be necessary to include a small quantity ofwater-miscible organic solvent such as DMSO to enhance thesolubilization of the components and complex formation. Once ananoparticulate gene carrier is formed, it is possible to remove thesolvent via centrifugal filtration or dialysis. It is possible that theincreased stability may interfere with intracellular dissociation ofcomplex. Should this be the case, a lipid may be grafted to thesecondary amine via disulfide bond, which can facilitate intracellulardissociation of the lipid. An amine derivative of bile acid may first bereacted with dithiobis [succinimidyl propionate] and then reacted withCLPEI in the presence of N,N-diisopropylethylamine.

Example 4: Treating a Chronic Liver Disease with Compositions of theInvention

By using various genetic, genomics and systems-based approach, a numberof polymorphisms, genes, lipids and miRNAs have been identified that aresignificantly associated with NAFLD and NASH susceptibility or drugresponse in treatment of NASH. Specifically for this application, it hasbeen discovered that PNPLA3 148M has a high baseline expression level inhuman liver due to the linkage disequilibrium of rs738409 G (encodingthe 148M isoform) and an intronic expression quantitative trait loci(eQTL) for PNPLA3, which confirms that a high transcription level ofPNPLA3 148M is related to an increased risk for NAFLD/NASH.

LCA-LPEI has been successfully synthesized as well as a ternary complexof pEGFP, LCA-LPEI, and DS at varying polymer/DNA weight ratios (10/1 to20/1 w/w). LCA-LPEI has showed greater gene transfection efficiency thanLPEI. DS increased transfection for both LPEI and LCA-LPEI polyplexes atall levels of polymer/DNA ratios (FIGS. 4A-C).

Transient co-transfection of 148MSi and PNPLA3-Luciferase (PNPLA3-Luc)vectors into HEK293 cells has demonstrated that the 148M but not the148I isoform was significantly down-regulated by 148MSi (FIGS. 3A-C andFIG. 7). This regulation is dose-dependent with a IC50 (50% of mRNA tobe cleared) of 8 pM (FIG. 3A). Further analyses demonstrated that 148MSisignificantly downregulates endogenous level of PNPLA3 148M mRNA (FIG.3B) but not the PNPLA3 148I wildtype (FIG. 3C). More importantly, 148MSitransient transfection significantly reduces the TG accumulation inHepG2 cells under induction by glucose (FIGS. 3D-E), as the baselinePNPLA3 expression in HepG2 is insufficient for inducing steatosis.148MSi as an allele-specific siRNA has outperformed the gene knockdowneffect of a commercial PNPLA3 siRNA set (Santa Cruz Biotechnology,Dallas, Tex., a mixture of multiple siRNA with unknown sequences) (FIGS.3B-C).

Example 5: Treating a Chronic Liver Disease In Vivo with Compositions ofthe Invention

Compositions of the invention (e.g., mut-specific siRNA) were tested invivo. The human PNPLA3^(148M) was transduced into mice (n=9) liver viatail-vein injection of adenovirus particles. After 1 week of virusinjection, siRNA or PBS (control) that was packed with INVIVOFECTAMINE3.0 (an animal origin-free lipid nanoparticle designed for highefficiency in vivo delivery of siRNA and miRNA to mouse liver cellsfollowing tail vein injection; Thermo Fisher Scientific, Waltham, Mass.)were injected into the mice. Mice were sacrificed after 2 weeks, thehuman PNPLA3 gene expression as well as total triglycerides (TG) levelsin the liver tissue were measured. FIG. 8A shows the significant(p<0.05) reduction of hepatic PNPLA3^(148M) expression after siRNAtreatment. FIGS. 8B through 8D demonstrated the significant (p<0.05)reduction of total hepatic TG levels after siRNA treatment.

What is claimed is:
 1. A composition comprising a small interfering RNA(siRNA) molecule that specifically downregulates expression of ars738409 C>G variant of a patatin-like phospholipase domain-containing(PNPLA3) gene, wherein the siRNA molecule comprises a nucleotidesequence of at least one of SEQ ID NOs 288-297.
 2. The compositionaccording to claim 1, further comprising a nanoparticle to which thesiRNA molecule is coupled.
 3. The composition according to claim 2,wherein the nanoparticle comprises low molecular weightpolyethyleneimine (LPEI) and a lipid.
 4. The composition according toclaim 3, wherein the lipid is a bile acid.
 5. The composition accordingto claim 1, wherein the siRNA molecule does not downregulate expressionof a wild-type isoform of the PNPLA3 gene.
 6. The composition accordingto claim 1, wherein the siRNA molecule comprises one or morenon-naturally occurring nucleotides.
 7. A method for treating a subjectwith a chronic liver disease or alcoholic liver disease (ALD), themethods comprising administrating a therapeutically effective amount ofa composition comprising a small interfering RNA (siRNA) molecule thatspecifically downregulates expression of a rs738409 C>G variant of apatatin-like phospholipase domain-containing (PNPLA3) gene, wherein thesiRNA molecule comprises a nucleotide sequence of at least one of SEQ IDNOs 288-297.
 8. The method according to claim 7, wherein the compositionfurther comprises a nanoparticle to which the siRNA molecule is coupled.9. The method according to claim 8, wherein the nanoparticle compriseslow molecular weight polyethyleneimine (LPEI) and a lipid.
 10. Themethod according to claim 9, wherein the lipid is a bile acid.
 11. Themethod according to claim 7, wherein the siRNA molecule comprises one ormore non-naturally occurring nucleotides.